Exercise promotes learning and memory formation. These effects depend on increases in hippocampal BDNF, a growth factor associated with cognitive improvement and the alleviation of depression symptoms. Identifying molecules that are produced during exercise and that mediate hippocampal Bdnf expression will allow us to harness the therapeutic potential of exercise. Here, we report that an endogenous molecule produced during exercise in male mice induces the Mus musculus Bdnf gene and promotes learning and memory formation. The metabolite lactate, which is released during exercise by the muscles, crosses the blood-brain barrier and induces Bdnf expression and TRKB signaling in the hippocampus. Indeed, we find that lactate-dependent increases in BDNF are associated with improved spatial learning and memory retention. The action of lactate is dependent on the activation of the Sirtuin1 deacetylase. SIRT1 increases the levels of the transcriptional coactivator PGC1a and the secreted molecule FNDC5, known to mediate Bdnf expression. These results reveal an endogenous mechanism to explain how physical exercise leads to the induction of BDNF, and identify lactate as a potential endogenous molecule that may have therapeutic value for CNS diseases in which BDNF signaling is disrupted.It is established that exercise promotes learning and memory formation and alleviates the symptoms of depression. These effects are mediated through inducing Bdnf expression and signaling in the hippocampus. Understanding how exercise induces Bdnf and identifying the molecules that mediate this induction will allow us to design therapeutic strategies that can mimic the effects of exercise on the brain, especially for patients with CNS disorders characterized by a decrease in Bdnf expression and who cannot exercise because of their conditions. We identify lactate as an endogenous metabolite that is produced during exercise, crosses the blood-brain barrier and promotes hippocampal dependent learning and memory in a BDNF-dependent manner. Our work identifies lactate as a component of the "exercise pill."

Macroautophagy (or autophagy) is a conserved degradative pathway that has been implicated in a number of biological processes, including organismal aging, innate immunity, and the progression of human cancers. This pathway was initially identified as a cellular response to nutrient deprivation and is essential for cell survival during these periods of starvation. Autophagy is highly regulated and is under the control of a number of signaling pathways, including the Tor pathway, that coordinate cell growth with nutrient availability. These pathways appear to target a complex of proteins that contains the Atg1 protein kinase. The data here show that autophagy in Saccharomyces cerevisiae is also controlled by the cAMP-dependent protein kinase (PKA) pathway. Elevated levels of PKA activity inhibited autophagy and inactivation of the PKA pathway was sufficient to induce a robust autophagy response. We show that in addition to Atg1, PKA directly phosphorylates Atg13, a conserved regulator of Atg1 kinase activity. This phosphorylation regulates Atg13 localization to the preautophagosomal structure, the nucleation site from which autophagy pathway transport intermediates are formed. Atg13 is also phosphorylated in a Tor-dependent manner, but these modifications appear to occur at positions distinct from the PKA phosphorylation sites identified here. In all, our data indicate that the PKA and Tor pathways function independently to control autophagy in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site of signal integration within this degradative pathway.cAMP-dependent protein kinase ͉ macroautophagy ͉ stationary phase ͉ Tor protein kinase M acroautophagy (hereafter autophagy) is a highlyconserved membrane trafficking pathway that is responsible for the turnover of bulk cytoplasmic protein and organelles (1, 2). This pathway was initially identified as a cellular response to nutrient deprivation (3, 4). However, recent studies indicate that autophagy is involved in a wide variety of physiological processes, including tissue remodeling during development, the removal of protein aggregates, and innate immune responses (5, 6). During autophagy, an isolation membrane emanates from a nucleation site that is known as the preautophagosomal structure (PAS) in Saccharomyces cerevisiae and the phagophore assembly site in mammals (7,8). This double membrane encapsulates nearby cytoplasm and ultimately targets it to the vacuole/ lysosome for degradation. The breakdown products are then recycled to allow for the synthesis of the macromolecules needed for survival during the period of starvation (9). The cellular components mediating autophagy were initially described in S. cerevisiae, and orthologs of many of these Atg proteins have since been identified in other eukaryotes (10, 11).The flux through the autophagy pathway is tightly controlled by multiple signaling pathways, including the Tor pathway, that are responsible for coordinating cell growth with nutrient availability. One of the key targets of this control a...

Protein kinases are important mediators of much of the signal transduction that occurs in eukaryotic cells. Unfortunately, the identification of protein kinase substrates has proven to be a difficult task, and we generally know few, if any, of the physiologically relevant targets of any particular kinase. Here, we describe a sequence-based approach that simplified this substrate identification process for the cAMP-dependent protein kinase (PKA) in Saccharomyces cerevisiae. In this method, the evolutionary conservation of all PKA consensus sites in the S. cerevisiae proteome was systematically assessed within a group of related yeasts. The basic premise was that a higher degree of conservation would identify those sites that are functional in vivo. This method identified 44 candidate PKA substrates, 5 of which had been described. A phosphorylation analysis showed that all of the identified candidates were phosphorylated by PKA and that the likelihood of phosphorylation was strongly correlated with the degree of target site conservation. Finally, as proof of principle, the activity of one particular target, Atg1, a key regulator of autophagy, was shown to be controlled by PKA phosphorylation in vivo. These data therefore suggest that this evolutionary proteomics approach identified a number of PKA substrates that had not been uncovered by other methods. Moreover, these data show how this approach could be generally used to identify the physiologically relevant occurrences of any protein motif identified in a eukaryotic proteome.Ras proteins ͉ sequence conservation ͉ stationary phase P rotein kinases are key components of signal transduction pathways that regulate many aspects of eukaryotic biology (1, 2). The protein kinase gene family is one of the largest in eukaryotic organisms and typically constitutes almost 2% of all protein-encoding genes (3, 4). In general, these enzymes catalyze the transfer of the terminal phosphate from ATP to the hydroxyl group of particular serine, threonine, or tyrosine residues in a defined set of protein targets. This phosphorylation ultimately alters cell physiology by modifying the activities associated with these substrate proteins. A complete understanding of the biology of any protein kinase therefore requires the identification of the particular substrates of this enzyme. Unfortunately, this identification process is often a difficult and labor-intensive task, and, as a result, we generally know few of the physiologically relevant substrates of any protein kinase (5).The cAMP-dependent protein kinase (PKA) has been extensively studied and is one of the best understood members of the protein kinase family (6, 7). In Saccharomyces cerevisiae, PKA is a key regulator of cell growth and is regulated largely by the small GTP-binding Ras proteins (8-10). The two Ras proteins, Ras1 and Ras2, bind to adenylyl cyclase and stimulate the production of cAMP (11,12). This stimulation results in elevated PKA activity and the increased phosphorylation of substrates that are presumably import...

Consolidation of long-term memories depends on de novo protein synthesis. Several translational regulators have been identified, and their contribution to the formation of memory has been assessed in the mouse hippocampus. None of them, however, has been implicated in the persistence of memory. Although persistence is a key feature of long-term memory, how this occurs, despite the rapid turnover of its molecular substrates, is poorly understood. Here we find that both memory storage and its underlying synaptic plasticity are mediated by the increase in level and in the aggregation of the prion-like translational regulator CPEB3 (cytoplasmic polyadenylation element-binding protein). Genetic ablation of CPEB3 impairs the maintenance of both hippocampal long-term potentiation and hippocampus-dependent spatial memory. We propose a model whereby persistence of long-term memory results from the assembly of CPEB3 into aggregates. These aggregates serve as functional prions and regulate local protein synthesis necessary for the maintenance of long-term memory.

When faced with nutrient deprivation, Saccharomyces cerevisiae cells enter into a nondividing resting state, known as stationary phase. The Ras/PKA (cAMPdependent protein kinase) signaling pathway plays an important role in regulating the entry into this resting state and the subsequent survival of stationary phase cells. The survival of these resting cells is also dependent upon autophagy, a membrane trafficking pathway that is induced upon nutrient deprivation. Autophagy is responsible for targeting bulk protein and other cytoplasmic constituents to the vacuolar compartment for their ultimate degradation. The data presented here demonstrate that the Ras/PKA signaling pathway inhibits an early step in autophagy because mutants with elevated levels of Ras/PKA activity fail to accumulate transport intermediates normally associated with this process. Quantitative assays indicate that these increased levels of Ras/PKA signaling activity result in an essentially complete block to autophagy. Interestingly, Ras/PKA activity also inhibited a related process, the cytoplasm to vacuole targeting (Cvt) pathway that is responsible for the delivery of a subset of vacuolar proteins in growing cells. These data therefore indicate that the Ras/PKA signaling pathway is not regulating a switch between the autophagy and Cvt modes of transport. Instead, it is more likely that this signaling pathway is controlling an activity that is required during the early stages of both of these membrane trafficking pathways. Finally, the data suggest that at least a portion of the Ras/PKA effects on stationary phase survival are the result of the regulation of autophagy activity by this signaling pathway.